Nodaviruses are small icosahedral viruses with bipartite, single-stranded, positive-sense RNA genomes. Containing only 4.5 kb, their genomes are among the smallest and simplest of all known animal viruses, yet they replicate up to 100-fold more abundantly than most other viral RNAs, and can do so in cells from vertebrates, insects, plants, and even yeast. Because of this, steps in nodavirus replication and assembly can be examined at levels of resolution that cannot yet be achieved with any other RNA viruses of animals. Advances in our understanding of the dynamic RNA-protein interactions involved in nodavirus replication and assembly can be confidently expected to illuminate studies of many medically important positive-strand RNA viruses. Moreover, because of the simplicity, robustness, and extraordinary amplifying power of the nodavirus RNA replicases, they are promising candidates for development as components of expression vectors for a wide range of applications. The work described in this proposal will focus on the basic mechanisms of replication and encapsidation of nodavirus RNAs. We will undertake detailed studies of these processes by combining genetic, biochemical, and structural approaches to examine the interactions of the viral RNAs with the RNA-dependent RNA polymerase during replication and with the capsid protein precursor during virus assembly. In the area of RNA replication, we will examine the structures and functions of nodavirus RNA replication complexes; the template roles of covalent homo- and heterodimers of the viral RNAs; how the subgenomic RNA is synthesized and how it coordinates the replication of the viral genome segments; the structure of the RdRp and its ability to replicate RNA in vitro; and the viral guanylyl- and methyltransferase activities involved in RNA capping. In the area of capsid assembly and structure, we will examine: the arrangement of RNAs 1 and 2 in virions of Pariacoto virus (PaV), a newlydescribed nodavirus in which a significant fraction of the viral RNA is visible at 3A resolution in virus particles; the properties, structures, and encapsidated RNAs of immature PaV provirions and virus-like particles of PaV; the design of RNAs to compete for encapsidation with PaV RNAs 1 and 2; the roles in virion assembly of specific domains in the PaV capsid proteins; and the creation of PaV particles that have different versions of the capsid protein in the different quasi-equivalent positions of the capsid.